The conjugation of proteins with ubiquitin involves the sequential action of proteins known as E1s (ubiquitin activating enzymes), E2s or UBCs (ubiquitin conjugating enzymes), and E3s (ubiquitin protein ligases). We have characterized a family of closely related E2s and gone on to establish that Nedd-4, which is related to E6-AP (the E3 that ubiquitinates p53 in the presence of human papilloma virus E6) in its carboxyl-terminus, is an enzymatically active E3. More recently we have utilized an E2 enzyme (UbcH5B) in the yeast two-hybrid system to isolate novel E3s. This has resulted in the isolation of cDNAs encoding a protein that binds UbcH5B and related E2s both in vivo and in vitro. This protein, tentatively designated AO7, is an enzymatically active E3. Structure/function analysis on this protein is currently ongoing. Other studies in this project have resulted in the determination that an E2 enzyme that is differentially expressed during neural development functions in conjunction with E6-AP but not with Nedd-4. In other studies, we have determined that degradation of the iron regulatory protein-2 in response to oxidation is mediated by the ubiquitin conjugating system, and that b-catenin is also degraded as a consequence of ubiquitination. Other ongoing studies, being carried out together with Dr. Michael Kuehn (EIB), have involved the use of Nedd-4 as bait in a yeast two-hybrid screen. This has resulted in the isolation of several proteins that both bind to, and are substrates for, ubiquitination by Nedd-4.